Primes and probes for the amplification and detection of aids associated nucleic acids

ABSTRACT

The presence or absence of a nucleic acid sequence associated with AIDS in a sample containing one or more nucleic acids and suspected of containing such sequence can be detected by amplifying the sequence using primers to form extension products as templates and detecting the amplified product if it is present. This may be accomplished by adding a labeled hybridization probe to the amplified product either free in solution or after immobilization on a solid support. Exemplary primers and probes for amplifying and detecting AIDS virus are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 07/918,907,filed Jul. 22, 1992, now abandoned, which is a continuation, ofapplication Ser. No. 07/639,103, filed Jan. 9, 1991, now abandoned,which is a continuation of U.S. Ser. No. 394,276filed Aug. 15, 1989,which issued as U.S. Pat. No. 5,0008,182, which is a continuation ofU.S. Ser. No. 935,581, filed Nov. 26, 1986, now abandoned, which is acontinuation-in-part application of U.S. Ser. No. 818,127, filed Jan.10, 1986, now abandoned. U.S. Ser. No. 394,276, is also acontinuation-in-part application of U.S. Ser. No. 828,144, filed Feb. 7,1986, which issued as U.S. Pat. No. 4,683,195 on Jul. 28, 1987, which isa continuation-in-part of U.S. Ser. No. 824,044, filed Jan. 30, 1986,now abandoned, which is a divisional of U.S. Ser. No. 791,308, filedOct. 25, 1985, which issued as U.S. Pat. No. 4,683,202 on Jul. 28, 1987,and which is a continuation-in-part of U.S. Ser. No. 716,975, filed Mar.28, 1985, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to a process for detecting the presence orabsence of a conserved, nucleotide sequence of a virus associated withacquired immune deficiency syndrome (AIDS). This invention also relatesto a kit for such detection having primers and a labeled hybridizationprobe.

The acquired immune deficiency syndrome (AIDS) is a transmissibledisorder of the cellular immune system resulting in frequently fatalopportunistic infections or neoplasms. In addition, AIDS is frequentlycomplicated by central nervous system dysfunction. The aetiologicagent(s) responsible for this disease has been identified as a humanretrovirus and designated as human T cell leukemia virus III (HTLVIII),lymphadenopathy associated virus (LAV or LAVA ), and AIDS-associatedvirus (ARV-2). More recently these viruses have been collectivelyreferred to as human immunodeficiency virus (HIV). The isolates from thevarious laboratories represent identical or closely related viruses bynumerous criteria (i.e., morphology, immunological cross-reactivities ofenvelope and nucleocapsid proteins, nucleotide sequence, and entry intohelper T cells using the T4 antigen). A simian virus isolated fromchimpanzees and macaques suffering from symptoms indistinguishable fromAIDS in humans is also closely related by these same criteria. P. J.Kanki et al., Science,230:951-954 (1985).

One of the more intriguing observations about the viruses associatedwith AIDS is their resemblance to the mature virion of subfamilyLentiviridae. Members of this pathogenic but non-oncogenic viral groupinclude visna virus, and equine infectious anemia virus. Thesimilarities between the AIDS-associated viruses and lentivirusesinclude virion morphology, immunological cross-reactivity, nucleotidesequence, brain localization, replication, .and marked heterogeneity.

The current immunodiagnostic tests to identify sera with antibodies tothe AIDS-associated virus(es) (see U.S. Pat. No. 4,520,113 to Gallo etal.) are being used in blood banks to eliminate potentially infectiousblood. See also WO 86/01834 published Mar. 27, 1986 (University ofCalifornia) for retroviral polypeptides useful in preparing monoclonalantibodies to detect retroviruses in the HTLV family. Because thesimilarities of the AIDS-associated viruses and lentiviruses, ingeneral, or visna specifically, may extend to the ability of thevirus(es) to reside as a DNA copy without producing significantquantities of viral particles, a direct immunological approach to detectAIDS-associated viruses may prove unsuccessful in a significant fractionof persistently infected asymptomatic individuals. Because the number ofvirus particles in the infected tissues and blood may be few (due toviral quiescence), direct detection of viral particles or RNA/DNA may bedifficult, if not impossible, without co-culturing the infected cellswith a permissive T cell line. Even with co-cultivation, the number ofindividuals infected by HIV as indicated by virus isolation is anunderestimate of the true number of infected individuals; virus fromonly 50% AIDS patients, 85% of ARC, and 30% of healthy individuals atrisk for AIDS was isolated (Salahuddin et al., PNAS USA, 82, 5530-4(1985)).

U.S. Pat. No. 4,683,202 by inventor K. Mullis describes a process foramplifying nucleic acid sequences to facilitate detection thereof, as byusing a labeled RNA or DNA hybridization probe. In this process primersare used to obtain primer extension products which are used as templatesto synthesize additional complementary strands in the presence ofnucleotides. The above-mentioned patent application also describes atechnique whereby after a probe is hybridized to the desired sequence, arestriction enzyme is added to cleave the hybrid at a site within thedesired sequence, and the restriction digest is then analyzed forlabeled fragments. U.S. Pat. No. 4,683,195 by inventors H. Erlich et al.and Saiki et al., Biotechnology,3:1008-1012 (1985) describe this lattertechnique in greater detail. Both patent applications illustrate use ofthe process for detecting genetic diseases such as sickle cell anemiaand β-thalassemia. These methods and the process for amplifying nucleicacid sequences are also disclosed in Saiki et al., Science,230,1350-1354 (1985), the disclosure of which is incorporated herein byreference. This application is related to U.S. Ser. No. 394,145, filedAug. 15, 1989, which is a continuation of U.S. Ser. No. 934,955, filedNov. 26, 1986, entitled, "Detection of Viruses by Amplification andHybridization," now abandoned, which is a continuation in part of818,127 filed Jan. 10, 1987, now abandoned.

A review article by Landry et al., Clin. Lab. Med. (1985) 5, 513-529describes the field of nucleic acid hybridization as applied to virusdetection. WO86/01535 published Mar. 13, 1986 and EP 173,529 publishedMar. 5, 1986 disclose molecular cloning of HTLVIII and use of the cloneas a probe to detect AIDS. Further, EP patent publication 173,339,published Mar. 5, 1986, discloses a genetic analysis using a DNA probeto detect infections by foreign microbes. EP 185,444, published Jun. 25,1986, discloses a recombinant peptide for use as a probe to detect theHTLVIII virus in cell lysates. Oncor Inc. announced in September, 1986that it has developed a radioactive blood test to detect the AIDS virus.

Use of a hybridization probe to detect AIDS may allow identification ofthose individuals who are persistently infected but are not producingvirus or individuals who are antibody negative but culture positive, andto detect infected cells without the need to culture the virus.Increasing the viral nucleic acid copy number of the virus byamplification will facilitate the identification of viral nucleic acidin infected individuals.

SUMMARY OF THE INVENTION

The present invention involves a process for detecting or monitoring forthe presence or absence of a nucleic acid sequence which issubstantially conserved among the nucleic acids in AIDS viruses andspecific to the nucleic acids in HIV (AIDS viruses) and which nucleicacid sequence is suspected of being contained in a sample, which processcomprises:

(a) treating the sample, together or separately, with an oligonucleotideprimer for each strand of the nucleic acid sequence, four differentnucleoside triphosphates, and an agent for polymerization, underhybridizing conditions, such that for each strand of the nucleic acidsequence an extension product of each primer is synthesized which iscomplementary to each nucleic acid strand, wherein said primer(s) aresubstantially complementary to each strand of the nucleic acid sequencebeing detected or monitored, such that the extension product synthesizedfrom one primer, when it is separated from its complement, can serve asa template for synthesis of the extension product of the other primer;

(b) treating the sample under denaturing conditions;

(c) treating the product of step (b) with oligonucleotide primers suchthat a primer extension product is synthesized using each of the singlestrands produced in step (b) as a template, resulting in amplificationof the specific nucleic acid sequence or sequences if present; and

(d) determining if the sequence to be detected is present in the sample.

One way to detect the product is by adding to the product of step (c) alabeled probe capable of hybridizing with the amplified nucleic acidsequence; and determining whether the probe has hybridized to anamplified sequence in the nucleic acid sample. In one embodiment, thisdetermination can be made by:

(1) digesting the hybridized mixture with a restriction enzymerecognizing a site within the sequences in the probe; and

(2) detecting whether the restriction digest contains a restrictionfragment correlated with the presence of the AIDS sequence.

Before step (a) the nucleic acids in a patient sample may be extractedtherefrom so that the sample being treated is actually the mixture ofthe extracted nucleic acids. In addition, the sample being treated instep (a) need not be subjected beforehand to a process wherein the virusin the sample is cultured.

In another embodiment, the invention herein relates to a kit fordetecting or monitoring for the presence or absence of a nucleic acidsequence which is substantially conserved among the nucleic acids inAIDS viruses and specific to the nucleic acids in AIDS viruses and whichnucleic acid sequence is suspected of being contained in a sample, whichkit comprises:

(a) one oligonucleotide primer for each strand of the nucleic acidsequence to be detected, which primer or primers are substantiallycomplementary to each strand of each specific nucleic acid sequence suchthat an extension product synthesized from one primer, when it isseparated from its complement, can serve as a template for the synthesisof the extension product of the other primer; and

(b) a labeled probe capable of hybridizing with the nucleic acidsequence.

Preferably, the kit also contains an agent for polymerization, fourdifferent nucleotides, and a means for detecting hybrids of the probeand sequence.

The test kit herein may be used in research tests, clinical tests andother diagnostic applications. In addition, it can be used to detectinfected cells without culturing the virus, a feature useful inmonitoring patients treated with various therapeutic agents to resolvethe infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the entire HIV genome, whichconsists of the long terminal repeat (LTR) noncoding regions and thegag, pol, env, Q (or sor) and F (or 3'ORF) coding regions, and showsfrom what region the primers were chosen for the examples herein.

FIG. 2 is a schematic representation of ten oligonucleotides of the HIVsequence which were ligated together to form a 180 bp DNA fragment for ahybridization probe to be used in dot blots to detect amplifiedproducts.

FIG. 3 is a schematic flow diagram of how two clones, M13mp10W:C7 andM13mp10W:D6, which contain nucleotide sequence alterations, were treatedto obtain a sequence identical to HIV. The resulting clone, M-13-GAG,contains the 180 bp insert for use as a hybridization probe in dotblots. In the FIG., X denotes mutation in the gene.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a process and kit for detecting ormonitoring for a nucleic acid sequence associated with HIV in a sampleof nucleic acid(s) suspected of containing the sequence. Because the HIVis highly variable, there is a need to find a common denominator(conserved region with a length which allows specific primers as definedherein to initiate polymerization) among the virus variants to use fordetecting a significant fraction of the viruses associated with AIDS. Asignificant fraction is that number of individuals sufficient to makethe test diagnostically or commercially feasible. Currently four HIVs,ARV, HTLVIII, LAV and LAVA, have been sequenced. These four initial HIVsand their associated variants are designated herein as "AIDS viruses".The sequence to be amplified also must be specific to the AIDS viruses,i.e., not react with HTLVI or HTLVII or other non-AIDS viruses.

The entire genome of the four HIV isolates and their variants isprovided by Sanchez-Pescador et al., Science,227,484-492 (1985) for ARV;Starcich et al., Science,227, 538-540 (1985) for HTLVIII; Wain-Hobson etal., Cell,9-17 (1985) for LAV; and Muesing et al., Nature,313, 450-458(1985) for LAVA. There is a general consensus that these viruses are allvariants of the same strain.

The term "substantially conserved" as applied to the sequence to bedetected signifies that the sequence must be sufficiently complementaryto the nucleic acids in the virus being detected to initiatepolymerization at least at room temperature in the presence of an agentfor polymerization and the four nucleoside triphosphates.

The primers used will be oligonucleotides of any length and sequence soas to provide specific initiation of polymerization on a significantnumber of nucleic acids in AIDS viruses. Specifically, the term "primer"as used herein refers to a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than three,which is capable of acting as a point of initiation of synthesis whenplaced under conditions in which synthesis of a primer extension productwhich is substantially complementary to a nucleic acid strand isinduced, i.e., in the presence of nucleoside triphosphates and an agentfor polymerization such as DNA polymerase and at a suitable temperatureand pH. The primer is preferably single stranded for maximum efficiencyin amplification, but may alternatively be double stranded. If doublestranded, the primer is first treated to separate its strands beforebeing used to prepare extension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent for polymerization. The exact lengths of the primers will dependon many factors, including temperature, buffer, nucleotide compositionand source of primer. For purposes herein, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides. Preferably the primer is from the gag region.

The primers herein are selected to be "substantially" complementary toeach strand of the specific sequence to be amplified. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands under conditions which allow the agent forpolymerization to perform, i.e., the primers have sufficientcomplementarity with the sequence of the strand to be amplified tohybridize therewith and thereby form a template for synthesis of theextension product of the other primer. Preferably, the primers haveexact complementarity with the strand.

One may select the sequence being amplified from among the regions thatare substantially conserved among the AIDS viruses. Therefore, theprimers and probes may be identified by any suitable means. This may bedone manually by comparing the regions of the published nucleic acidsequences of the four AIDS viral genomes. Another more convenient methodis to use a computer program to compare the sequences. For this purpose,a commercial program with the underlying computer algorithm supplied byNational Biomedical Research Foundation using a dot matrix may beconveniently employed. This program involves inputting the nucleic acidsequences of the AIDS viruses and defining a window size for base pairhomology. The program employs graphics to compare the sequences ondifferent axes, and a dot appears where there is at least substantialhomology. Preferably, the window size is greater than six bases.

A dot matrix program reveals that the gag region of the genome (see FIG.1), also known as the nucleocapsid gene, is most conserved among thecoding regions in the four variants. The next most conserved codingregion is the pol region, followed by the env region of the genome.Because gag is most conserved among the coding regions, it is thepreferred region from which to select primers and probes for detectingthe sequence. Regions of the viral genome that do not encode proteinscan also be used to determine a sequence for the primers to be used. Forpurposes herein, to maximize sensitivity and specificity, the sequencebeing detected is homologous with a sequence of a length sufficient toallow specific priming which is substantially conserved among therelated viruses, particularly at the restriction cleavage site if aprobe and restriction enzyme are employed.

The techniques used for amplifying and thereafter detecting the productare described in detail in U.S. Pat. Nos. 4,683,195 and 4,683,202, Saikiet al., Biotechnology, supra and Saiki et al., Science,supra, the entiredisclosures of which are incorporated herein by reference. In general,the amplification process involves an enzymatic chain reaction forpreparing, in exponential quantities relative to the number of reactionsteps involved, a specific nucleic acid sequence, given that the ends ofthe required sequence are known in sufficient detail thatoligonucleotide primers can be synthesized which will hybridize to them,and that a small amount of the sequence is available to initiate thechain reaction. One primer is complementary to the negative (-) strandand the other is complementary to the positive (+) strand. Annealing theprimers to denatured nucleic acid followed by extension with an enzymesuch as the large fragment of DNA Polymerase I (Klenow) and nucleotidesresults in newly synthesized + and - strands containing the targetsequence. Because these newly synthesized sequences are also templatesfor the primers, repeated cycles of denaturing,, primer annealing andextension results in exponential accumulation of the region defined bythe primer. The product of the chain reaction will be a discrete nucleicacid duplex with termini corresponding to the ends of the specificprimers employed.

The amplification process is illustrated diagrammatically below, wheredouble-stranded DNA containing the desired sequence [S] comprised ofcomplementary strands [S⁺ ] and [S⁻ ] is utilized as the nucleic acid.During the first and each subsequent reaction cycle extension of eacholigonucleotide primer on the original template will produce one newssDNA molecule product of indefinite length which terminates with onlyone of the primers. These products, hereafter referred to as "longproducts," will accumulate in a linear fashion; that is, the amountpresent after any number of cycles will be proportional to the number ofcycles.

The long products thus produced will act as templates for one or theother of the oligonucleotide primers during subsequent cycles and willproduce molecules of the desired sequence [S⁺ ] or [S⁻ ] These moleculeswill also function as templates for one or the other of theoligonucleotide primers, producing further [S⁺ ] and [S³¹ ], and thus achain reaction can be sustained which will result in the accumulation of[S] at an exponential rate relative to the number of cycles.

By-products formed by oligonucleotide hybridizations other than thoseintended are not self-catalytic and thus accumulate at a linear rate.

The specific sequence to be amplified, [S], can be depicteddiagrammatically as: ##STR1## The appropriate oligonucleotide primerswould be: ##STR2## so that if DNA containing [S] ##STR3## is separatedinto single strands and its single strands are hybridized to Primers 1and 2, the following extension reactions can be catalyzed by DNApolymerase in the presence of the four deoxyribonucleosidetriphosphates: ##STR4## On denaturation of the two duplexes formed, theproducts are: ##STR5## If these four strands are allowed to rehybridizewith Primers 1 and 2 in the next cycle, the agent for polymerizationwill catalyze the following reactions: ##STR6## If the strands of theabove four duplexes are separated, the following strands are found:##STR7##

It i s seen that each strand which terminates with the oligonucleotidesequence of one primer and the complementary sequence of the other isthe specific nucleic acid sequence [S] that is desired to be produced.

The steps of this process can be repeated indefinitely, being limitedonly by the amount of Primers 1 and 2, inducing agent and nucleotidespresent. The amount of original nucleic acid remains constant in theentire process, because it is not replicated. The amount of the longproducts increases linearly because they are produced only from theoriginal nucleic acid. The amount of the specific sequence increasesexponentially. Thus, the specific sequence will become the predominantspecies. This is illustrated in the following table, which indicates therelative amounts of the species theoretically present after n cycles,assuming 100% efficiency at each cycle:

    ______________________________________                                        Number of Double Strands After 0 to n Cycles                                                        Long       Specific                                     Cycle Number Template Products   Sequence [S]                                 ______________________________________                                        0            1        --         --                                           1            1        1          0                                            2            1        2          1                                            3            1        3          4                                            5            1        5          26                                           10           1        10         1013                                         15           1        15         32,752                                       20           1        20         1,048,555                                    n            1        n          (2.sup.n -n-1)                               ______________________________________                                    

When a single-stranded nucleic acid is utilized as the template, onlyone long product is formed per cycle.

As used herein, the terms "restriction endonucleases" and "restrictionenzymes" refer to bacterial enzymes of which cut double-stranded DNA ator near a specific nucleotide sequence.

The primer(s) herein may be selected by the following criteria, whichare factors to be considered, but are not exclusive or determinative.First, the primers are selected from conserved regions of the AIDSgenome. The gag region (nucleocapsid gene) is the most conserved of thecoding regions, followed by the pol and env regions, and therefore, thegag region was chosen for initial studies.

Secondly, the primer lacks homology with any sequences of vital genomesthat would be expected to compromise the test, those sequences forHTLVI, for example, being published by Seiki, M. et al., PNAS (USA)80:3618-3622 (1983).

Third, the primer preferably lacks secondary structure formation in theamplified nucleic acid which may interfere with extension by theamplification enzyme such as E. coli DNA polymerase, preferably thatportion of the DNA polymerase referred to as the Klenow fragment. Thismay be accomplished by employing up to about 15% by weight, preferably5-10% by weight, dimethyl sulfoxide (DMSO) in the amplification mediumand/or increasing the amplification temperatures to 30°-40° C.,preferably 35°-40° C.

Fourth, the primer preferably has an approximate 50% content of guanineand cytosine, and does not contain multiple consecutive adenine andthymine residues at the 3' end of the primer which may result in lessstable hybrids. Finally, if the amplified product will be detected byuse of a restriction enzyme, the probe must have an internal(non-terminal) restriction site.

The oligonucleotide primers may be prepared using any suitable method,such as, for example, the phosphotriester and phosphodiester methodsdescribed above, or automated embodiments thereof. In one such automatedembodiment diethylphosphoramidites are used as starting materials andmay be synthesized as described by Beaucage et al., Tetrahedron Letters(1981), 22:1859-1862. One method for synthesizing oligonucleotides on amodified solid support is described in U.S. Pat. No. 4,458,066. It isalso possible to use a primer which has been isolated from a biologicalsource (such as a restriction endonuclease digest).

Any source of nucleic acid, in purified or nonpurified form, can beutilized as the starting nucleic acid or acids, provided it contains oris suspected of containing the specific nucleic acid sequence associatedwith AIDS. Thus, the process may employ, for example, DNA or RNA,including messenger RNA, which DNA or RNA may be single stranded ordouble stranded. In the event that RNA is to be used as a template,enzymes and/or conditions optimal for reverse transcribing the templateto DNA would be utilized. In addition, a DNA-RNA hybrid which containsone strand of each may be utilized. A mixture of any of these nucleicacids may also be employed, or the nucleic acids produced from aprevious amplification reaction herein using the same or differentprimers may be so utilized. The specific nucleic acid sequence to beamplified may be only a fraction of a larger molecule or can be presentinitially as a discrete molecule, so that the specific sequenceconstitutes the entire nucleic acid. It is not necessary that thesequence to be amplified be present initially in a pure form; it may bea minor fraction of a complex mixture, such as a portion of thevirus-encoding gene contained in whole human DNA. The starting nucleicacid may contain more than one desired specific nucleic acid sequencewhich may be the same or different. Therefore, the present process isuseful not only for producing large amounts of one specific nucleic acidsequence, but also for amplifying simultaneously more than one differentspecific nucleic acid sequence located on the same or different nucleicacid molecules.

The nucleic acid(s) may be obtained from any source, for example,natural DNA or RNA from higher organisms such as animals. DNA or RNA maybe extracted from a bodily sample, such as blood, tissue material suchas chorionic villi, or amniotic cells by a variety of techniques such asthat described by Maniatis et al., Molecular Cloning (1982), 280-281.

If the sample is impure such as plasma, serum or blood, beforeamplification it may be treated with an amount of a reagent effective toopen the cells, fluids, tissues, viral capsids or animal cell membranesof the sample, and to expose and/or separate the strand(s) of thenucleic acid(s). This lysing and nucleic acid denaturing step to exposeand separate the strands will allow amplification to occur much morereadily. In addition, the AIDS virus need not be cultivated in thesample before the sample is treated with the amplification reagents. Thesample may be centrifuged to obtain buffy coats, which are then passedthrough a column to obtain leukocytes. The leukocytes may then betreated to extract the nucleic acids therefrom for use as the sample tobe amplified.

Any specific nucleic acid sequence can be produced by the presentprocess. It is only necessary that a sufficient number of bases at bothends of the sequence be known in sufficient detail so that twooligonucleotide primers can be prepared which will hybridize todifferent strands of the desired sequence and at relative positionsalong the sequence such that an extension product synthesized from oneprimer, when it is separated from its template (complement), can serveas a template for extension of the other primer into a nucleic acid ofdefined length. The greater the knowledge about the bases at both endsof the sequence, the greater can be the specificity of the primers forthe target nucleic acid sequence, and thus the greater the efficiency ofthe process. It will be understood that the word primer as usedhereinafter may refer to more than one primer, particularly in the casewhere there is some ambiguity in the information regarding the terminalsequence(s) of the fragment to be amplified. For instance, in the casewhere a nucleic acid sequence is inferred from protein sequenceinformation a collection of primers containing sequences representingall possible codon variations based on degeneracy of the genetic codewill be used for each strand. One primer from this collection will besubstantially conserved with the end of the desired sequence to beamplified.

The specific nucleic acid sequence is produced by using the nucleic acidcontaining that sequence as a template. If the target nucleic acidsequence of the sample contains two strands, it is necessary to separatethe strands of the nucleic acid before it can be used as the template,either as a separate step or simultaneously with the synthesis of theprimer extension products. This strand separation can be accomplishedusing any suitable denaturing conditions, including physical, chemicalor enzymatic means, the word "denaturing" used herein to include allsuch means. One physical method of separating the strands of the nucleicacid involves heating the nucleic acid until it is denatured. Typicalheat denaturation may involve temperatures ranging from about 80° to105° C. for times ranging from about 1 to 10 minutes. Strand separationmay also be induced by an enzyme from the class of enzymes known ashelicases or the enzyme RecA, which has helicase activity and in thepresence of riboATP is known to denature DNA. The reaction conditionssuitable for separating the strands of nucleic acids with helicases aredescribed by Kuhn Hoffmann-Berling, CSH-Quantitative Biology, 43:63(1978), and techniques for using RecA are reviewed in C. Radding, Ann.Rev. Genetics 16:405-37 (1982).

If the original nucleic acid containing the sequence to be amplified issingle stranded, its complement is synthesized by adding one or twooligonucleotide primers thereto. If an appropriate single primer isadded, a primer extension product is synthesized in the presence of theprimer, an agent for polymerization, and the four nucleosidetriphosphates described below. The product will be partiallycomplementary to the single-stranded nucleic acid and will hybridizewith the nucleic acid strand to form a duplex of unequal length strandsthat may then be separated into single strands as described above toproduce two single separated complementary strands. Alternatively, twoappropriate primers may be added to the single-stranded nucleic acid andthe reaction carried out.

If the original nucleic acid constitutes the sequence to be amplified,the primer extension product(s) produced will be completely orsubstantially complementary to the strands of the original nucleic acidand will hybridize therewith to form a duplex of equal length strands tobe separated into single-stranded molecules.

When the complementary strands of the nucleic acid or acids areseparated, whether the nucleic acid was originally double or singlestranded, the strands are ready to be used as a template for thesynthesis of additional nucleic acid strands. This synthesis isperformed under conditions allowing hybridization of primers totemplates to occur. Generally it occurs in a buffered aqueous solution,preferably at a pH of 7-9, most preferably about 8. Preferably, a molarexcess (for genomic nucleic acid, usually about 10⁸ :1 primer:template)of the two oligonucleotide primers is added to the buffer containing theseparated template strands. It is understood, however, that the amountof complementary strand may not be known if the process herein is usedfor diagnostic applications, so that the amount of primer relative tothe amount of complementary strand cannot be determined with certainty.As a practical matter, however, the amount of primer added willgenerally be in molar excess over the amount of complementary strand(template) when the sequence to be amplified is contained in a mixtureof complicated long-chain nucleic acid strands. A large molar excess ispreferred to improve the efficiency of the process.

The deoxyribonucleoside triphosphates dATP, dCTP, dGTP and TTP are alsoadded to the synthesis mixture, either separately or together with theprimers, in adequate amounts and the resulting solution is heated toabout 90°-100° C. for from about 1 to 10 minutes, preferably from 1 to 4minutes. After this heating period the solution is allowed to cool toroom temperature, which is preferable for the primer hybridization. Tothe cooled mixture is added an appropriate agent for effecting theprimer extension reaction (called herein "agent for polymerization"),and the reaction is allowed to occur under conditions known in the art.The agent for polymerization may also be added together with the otherreagents if it is heat stable. This synthesis reaction may occur at fromroom temperature up to a temperature above which the agent forpolymerization no longer functions. Thus, for example, if DNA polymeraseis used as the agent, the temperature is generally no greater than about40° C. Most conveniently the reaction occurs at room temperature.

The agent for polymerization may be any compound or system which-willfunction to accomplish the synthesis of primer extension products,including enzymes. Suitable enzymes for this purpose include, forexample, E. coli DNA polymerase I, Klenow fragment of E. coli DNApolymerase I, T4 DNA polymerase, other available DNA polymerases,polymerase muteins, reverse transcriptase, and other enzymes, includingheat-stable enzymes (i.e., those enzymes which perform primer extensionafter being subjected to temperatures sufficiently elevated to causedenaturation), which will facilitate combination of the nucleotides inthe proper manner to form the primer extension products which arecomplementary to each nucleic acid strand. Generally, the synthesis willbe initiated at the 3' end of each primer and proceed in the 5'direction along the template strand, until synthesis terminates,producing molecules of different lengths. There may be agents forpolymerization, however, which initiate synthesis at the 5' end andproceed in the other direction, using the same process as describedabove.

The newly synthesized strand and its complementary nucleic acid strandwill form a double-stranded molecule under the hybridizing conditionsdescribed above if the target sequence is present, and this hybrid isused in the succeeding steps of the process. In the next step, thesample treated under hybridizing conditions is subjected to denaturingconditions using any of the procedures described above to providesingle-stranded molecules if the target sequence is present.

New nucleic acid is synthesized on the single-stranded molecules.Additional agent for polymerization, nucleotides and primers may beadded if necessary for the reaction to proceed under the conditionsprescribed above. Again, the synthesis will be initiated at one end ofeach of the oligonucleotide primers and will proceed along the singlestrands of the template to produce additional nucleic acid. After thisstep, half of the extension product will consist of the specific nucleicacid sequence bounded by the two primers.

The steps of denaturing and extension product synthesis can be repeatedas often as needed to amplify the target nucleic acid sequence to theextent necessary for detection. As will be described in further detailbelow, the amount of the specific nucleic acid sequence produced willaccumulate in an exponential fashion.

When it is desired to produce more than one specific nucleic acidsequence from the first nucleic acid or mixture of nucleic acids, theappropriate number of different oligonucleotide primers are utilized.For example, if two different specific nucleic acid sequences are to beproduced, four primers are utilized. Two of the primers are specific forone of the specific nucleic acid sequences and the other two primers arespecific for the second specific nucleic acid sequence. In this manner,each of the two different specific sequences can be producedexponentially by the present process.

The present invention can be performed in a step-wise fashion whereafter each step new reagents are added, or simultaneously, where allreagents are added at the initial step, or partially step-wise andpartially simultaneous, where fresh reagent is added after a givennumber of steps. If a method of denaturation, such as heat, is employedwhich will inactivate the agent for polymerization, as in the case of aheat-labile enzyme, then it is necessary to replenish the agent afterevery strand separation step. The simultaneous method may be utilizedwhen an enzymatic means is used for the strand separation step. In thesimultaneous procedure, the reaction mixture may contain, in addition tothe nucleic acid strand(s) containing the desired sequence, thestrand-separating enzyme (e.g., helicase), an appropriate energy sourcefor the strand-separating enzyme, such as rATP, the four nucleosidetriphosphates, the oligonucleotide primers in molar excess, and theagent for polymerization, e.g., Klenow fragment of E. coli DNApolymerase I.

If heat is used for denaturation in a simultaneous process, aheat-stable agent such as a thermostable polymerase may be employedwhich will operate at an elevated temperature, preferably 50°-105° C.depending on the agent, at which temperature the nucleic acid willconsist of single and double strands in equilibrium. For smaller lengthsof nucleic acid, lower temperatures of about 40°-50° C. may be employed.The upper temperature will depend on the temperature at which the enzymewill degrade or the temperature above which an insufficient level ofprimer hybridization will occur. Such a heat-stable enzyme is described,e.g., by A. S. Kaledin et al., Biokhimiya, 45, 644-651 (1980). For thisconstant temperature reaction to succeed, the primers have their 3' endswithin 6-8 base pairs of each other. Each step of the process will occursequentially notwithstanding the initial presence of all the reagents.Additional materials may be added as necessary. After the appropriatelength of time has passed to produce the desired amount of the specificnucleic acid sequence, the reaction may be halted by inactivating theenzymes in any known manner or separating the components of thereaction.

The amplification may also be carried out using a temperature-cyclingreaction wherein the temperature is increased incrementally to allow forextension, annealing and denaturation using a heat-stable enzyme. Thisprocess and the enzyme and the instrument that can be used therefor aredescribed more fully in copending U.S. application Ser. No. 899,513filed Aug. 22, 1986, now abandoned in favor of Ser. No. 063,647, filedJun. 17, 1987, which issued as U.S. Pat. No. 4,965,188, U.S. Pat. No.899,241 filed Aug. 22, 1986, and 899,061 filed Aug. 22, 1986, nowabandoned in favor of Ser. No. 063,509, which issued as U.S. Pat. No.4,889,818.

The process of the present invention may be conducted continuously. Inone embodiment of an automated process, the reaction may be cycledthrough a denaturing region, a reagent addition region, and a reactionregion. In another embodiment, the enzyme used for the synthesis ofprimer extension products can be immobilized in a column. The otherreaction components can be continuously circulated by a pump through thecolumn and a heating coil in series, thus the nucleic acids produced canbe repeatedly denatured without inactivating the enzyme.

The amplified product may be detected by analyzing it by Southern blotswithout using radioactive probes. In such a process, for example, asmall sample of DNA from, e.g., peripheral blood lymphocytes containinga very low level of the sequence associated with AIDS is amplified, andanalyzed via a Southern blotting technique. The use of non-radioactiveprobes is facilitated by the high level of the amplified signal.

Another method of detection involves detection using a labeled probecapable of hybridizing with the amplified nucleic acid sequence anddetermining if the probe has hybridized. Such probe necessarily containsa substantially conserved nucleic acid sequence from the genome of anAIDS virus (HTLVIII, ARV, LAY, LAVA, or a variant thereof) and isselected as described above for primers and amplified sequences.Preferably the probe is selected from the gag region of the AIDS genome.

One such probe method involves the oligomer restriction techniquedescribed in U.S. Pat. No. 4,683,194. In this procedure, the amplifiednucleic acid is denatured and hybridized in solution to a labeledoligonucleotide probe which hybridizes specifically to the targetsequence (spans the particular conserved region contained by theprimers) and spans at least one restriction site of interest. The duplexformed between target land probe will reconstitute the restriction site,and when cleaved with restriction enzyme, such as, e.g., BstNI, PvuII,or DraI, releases a labeled probe fragment which can he resolved fromthe full-length probe by gel electrophoresis. The resulting gel is thenautoradiographed. Analysis of the amplified product by this method israpid, i.e., results can be obtained in a few hours. Preferably, theprobe is 30-45 bases long and is labeled. Also, preferably therestriction enzyme is BstNI or PvuII.

Another method which may be used to analyze the amplified product is thedot blot method. In this method, the amplified samples are spotteddirectly on a membrane and hybridized with a labeled probe. The labelmay be detected by spectroscopy, photochemistry or by biochemical,immunochemical or chemical means. Examples include enzymes such asalkaline phosphatase, a radioactive label such as ³² p, a fluorescentlabel, or biotin. In one embodiment, the probe is a biotinylated probein which the biotin is attached to a spacer arm of the formula: ##STR8##where Y is O, NH or N--CHO, x is a number from 1 to 4, and y is a numberfrom 2 to 4. The spacer arm is in turn attached to a psoralen moiety ofthe formula: ##STR9## The psoralen moiety intercalates into andcrosslinks a "gapped circle" probe as described by Courage-Tebbe et al.,Biochim. Biophys. Acta, 697 (1982) 1-5, wherein the single-strandedhybridization region of the gapped circle spans the region containedbetween the primers. The details of this biotinylation and dot blotprocedure are described more fully in commonly assigned U.S. Pat. Nos.4,582,789 and 4,617,261, the disclosures of which are incorporatedherein by reference. The biotinylated probes eliminate the need forradioactive isotopes.

Alternatively, the probe may be spotted on the membrane first underprehybridization conditions if necessary and then the amplified productis added to the pre-treated membrane under hybridization conditions, "ina reverse" dot blot format.

The dot blot procedure is more time-consuming than the oligomerrestriction method described above, because the membrane must first beprehybridized and then hybridized with the probe. However, with rapidlymutating viruses, it has the advantage that sequences containing limitedbase mismatches are still detected under appropriate hybridizingconditions, whereas with the oligomer restriction method, any virusharboring a mutation which results in the abolishment of the restrictionsite will not be detected due to the variability of the virus.

The invention herein also contemplates a kit format which comprises apackaged multicontainer unit having containers of each primer and theprobe utilized. The kit may also have a container with the agent forpolymerization to synthesize the primer extension products, such asenzymes, a container with each of the four nucleoside triphosphates, anda container with means to detect the label (such as an avidin-enzymecomplex if the label is biotin). In addition, the kit may have acontainer which includes a positive control containing one or morenucleic acids with a sequence of the AIDS vital genome and/or acontainer including a negative control without such nucleic acids.Moreover, the kit may have a container for each restriction enzymecapable of cleaving a nucleic acid containing the target sequence at asite contained in a sequence in the probe.

The following examples illustrate various embodiments of the inventionand are not intended to be limiting in any respect. In the examples allparts and percentages are by weight if solid and by volume if liquid andall temperatures are in degrees Centigrade, unless otherwise indicated.

EXAMPLE 1

The desired sequences to be amplified were contained in eleven coded DNAsamples obtained from Dr. Bernard Poiesz of the Regional OncologyCenter, SUNY Upstate Medical Center, Syracuse, N.Y. 13210, identified as194 BK, 342, 367,361, 368H, 207, 307,308B, 323, 326 and 340. Thesequences to be amplified, the primers, and the probes were identifiedby the dot matrix program as described above, wherein the sequencewindow selected was at least 20 base pairs long, so that the sequenceswere chosen within conserved regions of the AIDS viruses.

The coded samples were first cultured in the presence of interleukin-2by Dr. Poeisz to test for the presence of virus. Then, the DNA wasextracted from the samples by the following procedure:

1. 1-2×10⁸ cultured cells were lysed in tubes with 20 ml of sodiumdodecyl sulfate lysis buffer (1% SDS, 150 mM NaCl, 25 mM Na₂ EDTA).

2. 400 μl of a 5 mg/μl solution of proteinase K was added per tube andincubated at 37° C. overnight.

3. The DNA was sequentially extracted with phenol, and CHCl₃ :isoamylalcohol followed by precipitation with ethanol.

4. The DNA was spooled out on a glass rod and resuspended in 1×TE buffer(10 nM Tris, 1 mM Na₂ EDTA, pH 7.5) and dialyzed exhaustively against1×TE buffer.

I. Synthesis of Primers

The following two oligodeoxyribonucleotide primers, designated SK01 andSK02, respectively, were prepared by the method described below:

    5'-CAGGGAGCTAGAACGAT-3'                                    (SK32)

    5'-CTTCTGATCCTGTCTGA-3'                                    (SK33)

(SK01 and SK02 were selected to provide for amplification of 107 basesbetween nucleotides 900 and 1006 of HTLVIII-isolate BH10.)

A. Automated Synthesis Procedures: The diethylphosphoramidites,synthesized according to Beaucage and Caruthers (Tetrahedron Letters(1981) 22:1859-1862) were sequentially condensed to a nucleosidederivatized controlled pore glass support using a Biosearch SAM-1. Theprocedure included detritylation with trichloroacetic acid indichloromethane, condensation using benzotriazole as activating protondonor, and capping with acetic anhydride and dimethylaminopyridine intetrahydrofuran and pyridine. Cycle time was approximately 30 minutes.Yields at each step were essentially quantitative and were determined bycollection and spectroscopic examination of the dimethoxytrityl alcoholreleased during detritylation.

B. Oligodeoxyribonucleotide Deprotection and Purification Procedures:The solid support was removed from the column and exposed to 1 mlconcentrated ammonium hydroxide at room temperature for four hours in aclosed tube. The support was then removed by filtration and the solutioncontaining the partially protected oligodeoxynucleotide was brought to55° C. for five hours. Ammonia was removed and the residue was appliedto a preparative polyacrylamide gel. Electrophoresis was carried out at30 volts/cm for 90 minutes after which the band containing the productwas identified by UV shadowing of a fluorescent plate. The band wasexcised and eluted with 1 ml distilled water overnight at 4° C. Thissolution was applied to an Altech RP18 column and eluted with a 7-13%gradient of acetonitrile in 1% ammonium acetate buffer at pH 6.0. Theelution was monitored by UV absorbance at 260 nm and the appropriatefraction collected, quantitated by UV absorbance in a fixed volume andevaporated to dryness at room temperature in a vacuum centrifuge.

C. Characterization of oligodeoxyribonucleotides: Test aliquots of thepurified oligonucleotides were ³² p labeled with polynucleotide kinaseand γ-³² P-ATP. The labeled compounds were examined by autoradiographyof 14-20% polyacrylamide gels after electrophoresis for 45 minutes at 50volts/cm. This procedure verifies the molecular weight. Base compositionwas determined by digestion of the oligodeoxyribonucleotide tonucleosides by use of venom diesterase and bacterial alkalinephosphatase and subsequent separation and quantitation of the derivednucleosides using a reverse phase HPLC column and a 10% acetonitrile, 1%ammonium acetate mobile phase.

II. Amplification Reaction

One microgram of DNA from each of the eleven coded DNA samples from Dr.Poiesz was added to 100 μl of buffer consisting of 10 mM Tris-HCl, pH7.5, 50 nM sodium chloride and 10 nM magnesium chloride and containing100 picomoles of Primer SK01, 100 picomoles of Primer SK02, and 150nanomoles each of dATP, dCTP, dGTP and TTP.

The resulting solution was heated to 100° C. for 10 minutes and allowedto cool to room temperature for two minutes, whereupon 2 μl containingone unit of Klenow fragment of E. coli DNA polymerase was added. Thereaction was allowed to proceed for two minutes at room temperature,after which the enzyme was inactivated by heating at 95° C. for twominutes. The denaturation, primer annealing, and extension with Klenow,two minutes per step, and adding polymerase were repeated nineteentimes.

III. Synthesis and Phosphorylation of Oligodeoxyribonucleotide Probe

A labeled DNA probe, SK03, of the sequence:

    5-*AATCCTGGCCTGTTAGAAACATCAGAAG-3',

where * indicates the label, was synthesized according to the proceduresdescribed in Section I. The probe was labeled by contacting 10 pmolethereof with 4 units of T4 polynucleotide kinase (New England Biolabs)and 50 pmole γ³² P-ATP (New England Nuclear, about 7200 Ci/mmole) in a40 μl reaction volume containing 70 mM Tris buffer (pH 7.6), 10 nMMgCl₂, 1.5 nM spermine, and 2.5 mM dithiothreitol for 90 minutes at 37°C. The total volume was then adjusted to 100 μl with 25 mM EDTA and analiquot removed for determination of specific activity by TCAprecipitation. The labeled probe was concentrated using Speed-vac andpurified by electrophoresis on a 18% polyacrylamide gel (19:1acrylamide:BIS,. Bio-Rad) in Trisboric acid-EDTA (TBE) buffer (89 nMTris, 89 mM boric acid, 2.5 mM EDTA, pH 8.3) at 500 volts for one hour.After localization by autoradiography, the portion of the gel containingthe labeled probe was excised, crushed and eluted into 0.2 ml TE bufferovernight at 4° C. TCA precipitation of the reaction product indicatedthat the specific activity was 2 Ci/mmole and the final concentrationwas 20 pmole/ml. IV. Hybridization/Digestion of Amplified Genomic DNAwith Probe and BstNI

Ten microliters of amplified DNA (containing the preamplificationequivalent of 71 μg of genomic DNA) was dispensed into a 1.5 mlMicrofuge tube and 20 μl of TE buffer to a final volume of 30 μl. Thesample was denatured at 95° C. for 10 minutes. Ten microliters of 0.6 MNaCl containing 0.02 pmole of SK03 probe was added to the tube, mixedgently, overlayed with mineral oil, and immediately transferred to a 56°C. heat block for one hour. Ten microliters of 50 nM MgCl₂ and 1 μl ofBstNI (10 units, New England Biolabs) were added and the reannealed DNAwas digested for 30 minutes at 56° C. The reaction was stopped by adding4 μl 75 mM EDTA and 6 μl tracking dye to a final volume of 60 μl.

The mineral oil was extracted with 0.2 ml chloroform, and 13 μl of thereaction mixture (˜15 ng genomic DNA) was loaded onto a 30%polyacrylamide mini-gel (19:1, Bio-Rad) in a Hoeffer SE200 apparatus.The gel was electrophoresed at approximately 300 volts for one houruntil the bromphenol blue dye front migrated to 3.0 cm off origin. Thetop 1.5 cm of the gel was removed and the remaining gel was exposed atleast overnight with two intensification screens at -70° C.

V. Discussion of Results

The autoradiograph showed that the AIDS DNA sequence was only present insample 368H, which was later found to be the only HTLVIII positive DNA.The other ten samples were as follows: (a) 194BK=DNA from leukemiapatient (no virus isolated), (b) 342=HTLVI, (c) 367=HTLVI, (d) 361HTLVI, (e) 207=patient with aggressive leukemia (skin involvement), (f)307=HTLVI prototype cell line (highest vital DNA to date), (g)308=HTLVI, (h) 323=HTLVII, (i) 326=HTLVI, and (j) 340=patient withaggressive leukemia (different from (e)).

Therefore, the primers employed were able to amplify the DNA to allowthe probe to detect accurately the sequence. The other sables remainednegative even with ten additional cycles of amplification. Amplificationin the presence of 10% DMSO (minimizes secondary structure formation) at37° C. also indicated the HTLVIII sample as the only positive sample.

EXAMPLE 2

In this example, the same procedure was followed as described in Example1 except that the primers employed, designated SK24 and SK18, were asfollows:

    5'-ATCCCAGTAGGAGAA-3'                                      (SK24)

    5'-TTATGTCCAGAATGC-3'                                      (SK18)

An alternative to SK24 was the primer SK25 as follows:

    5'-ATAATCCACCTATCCCAG-3'                                   (SK25)

The probe employed, SK19, was of the sequence:

    5'-*ATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTAC-3',

where * indicates the label. The probe was labeled as described inSection III. SK24 and SK18 were selected to provide for theamplification of a hydrophilic region of gag from nucleotides 1552 to1642 of HTLVIII. SK25 and SK18 were selected to provide for amplifying ahydrophilic region of gag from nucleotides 1541 to 1642 of HTLVIII.SK19, when annealed to the amplified DNA, reconstitutes a BstNI site.Digestion with this enzyme releases a 4-mer.

The autoradiograph for hybridization and restriction at 53° C. showed aspositive only the HTLVIII sample found in Example 1. A background bandappearing in all samples disappeared when the temperature ofhybridization and restriction was raised to 60° C. from 53° C. Theincreased temperature is presumed to have minimized nonspecifichybridization of the probe.

EXAMPLE 3

A 180 bp DNA fragment which encodes a hydrophilic region of gag(nucleotides 1470-1649 of HTLVIII-isolate BH10), having 186 base pairsincluding BamHI ends, was constructed by first ligating 10 overlappingoligomers using T4 DNA ligase. The oligomers are shown in FIG. 2. Theresulting fragment was then cloned into the BamHI site of M13mp10w whichis commercially available. None of the clones sequenced had the exactdesired sequence. However, using two clones shown in FIG. 3 (where Xdenotes a mutation in the gene), a clone with the correct sequence wasconstructed by substituting the SpeI/BstXI fragment of clone M13mp10:C7with the SpeI/BstXI fragment of clone M13mp10W:D6. DNA from M13mp10W:C7was digested with SpeI and BstXI, treated with alkaline phosphatase andthe larger vector containing fragment purified from an agarose gel. TheSpeI/BstXI DNA fragment from clone M13mp10W:D6 was purified from apolyacrylamide gel after double digestions with the same enzymes. Thepurified fragments from M13mp10W:C7 and M13mp10W:D6 were ligated andtransformed into E. coli strain DG98 available from the American TypeCulture Collection. The resulting clone, designated M-13-GAG, containsthe correct sequence and was deposited with the ATCC on Jan. 8, 1986with ATCC No. 40,218.

This M-13-GAG may be used to construct a gapped circle probe asdescribed above to evaluate an amplified sample in a non-isotopic dotblot format.

The amplified product may be prepared from two primers, SK23 and SK28,which encompass the entire 180 mer in M-13-GAG and have the followingsequence:

    5'-ATGAGAGAACCAAGG-3'                                      (SK23)

    5'-CCTTGTCTTATGTCCAG-3'                                    (SK28)

These primers, which were selected to amplify between nucleotides 1468and 1649, were already tested with probe SK19 and found to detect theHTLVIII sample successfully.

EXAMPLE 4

Seventy-one coded samples, the DNA of which was extracted by Dr. Poieszusing the method described in Example 2 were initially analyzed by theirDNA using the primer pair of SK01 and SK02 of Example 1 or SK17 andSK18, where SK18 is defined in Example 2 and SK17 has the sequence:

    5'-CCAGTAGGAGAAAT-3'

which is selected to amplify the hydrophilic region of gag fromnucleotides 1555 to 1642 of HTLVIII. The amplification using the primerpair SK17 and SK18 was carried out in the presence of 10% DMSO by weightat 37° C., but otherwise according to the procedure of Example 1. Afteramplification the procedure of Example 1 was used to detect the DNA,using the probe SK03 (of Example 1) or SK19 (of Example 2). Some of theambiguous samples were further analyzed using the primer pair SK24 andSK18 of Example 2 and using the probe SK19 at room temperature.

The results show that all samples which were identified as positive bythe test herein were DNAs isolated from AIDS or ARC patients, includinga HTLVIII isolate, a LAV isolate and an AIDS-associated virus (AAV)identified by Dr. Poiesz. In addition, an antibody positive, reversetranscriptase negative, healthy homosexual who has had multiple contactswith AIDS victims was also identified as positive by both sets of primerpairs. The SK17-SK18 and SK24-SK18 primer pairs appeared to detect morepositives than the SK01-SK02 primer pal r.

None of the negative control samples (normal T cells, uninfected celllines or HTLVII) showed positively in the assays herein. The test hereinidentified ten infected samples as positive which were negative byreverse transcriptase. On the other hand, five samples which werereverse transcriptase positive (3 out of the 5 were ±) were negative bythe test herein. All 71 samples proved to be ELISA .positive, showingthat ELISA is not a very discriminative or specific test for the AIDSvirus.

The above examples show that AIDS vital DNA sequences can be identifiedin cell lines infected with blood, semen mononuclear cells, and semensupernatants from patients with AIDS or ARC.

EXAMPLE 5

This example illustrates that the technique herein can be applieddirectly to the identification of AIDS vital DNA sequences in peripheralmononuclear cells of fresh blood without having to cultivate the virusfirst.

Coded blood samples from AIDS patients were treated as follows by Dr.Poeisz: First, they were centrifuged using low-speed centrifugation(about 300× g) to pellet all the cells, thereby obtaining bully coats.The bully coats were passed through a Ficoll-Hypaque density column andthe leukocytes were collected from the column. The DNA was extractedfrom the leukocytes by the procedure described in Example 1.

The DNA was amplified using the primer pairs SK17 and SK18 described inExamples 2 and 4, respectively, in the presence of 10% DMSO by weight at37° C. using the procedure described in Example 1 with 1 unit, 2 unitsand 4 units of Klenow fragment. After amplification, the probes used,following the procedure of Example 1, were either SK03 (of Example 1) orSK19 (of Example 2). Some of the ambiguous samples were further analyzedusing the primer pair SK24 and SK18 of Example 2 and the probe SK19 atroom temperature.

The results after overnight exposure show that the DNAs isolated fromsome of the AIDS or ARC patients were identified as positive.

The experiment was repeated using primer pairs SK23 and SK28 (describedin Example 3), and probe SK19 (Example 2). The experiment was againrepeated using primer pairs SK32 and SK33 having the sequences asfollows:

    5'-ACCTGCCACCTGTAGTAG-3'                                   (SK32)

    5'-GCCATATTCCTGGACTACAG-3'                                 (SK33)

and using the probe SK34 of the sequence:

    4'-TAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGAGAAGCC-3'            (SK34)

The restriction enzyme PvuII was used to cleave the restriction site ofSK34.

The results from both experiments after a 6-day exposure period revealedthat the DNAs isolated from some of the AIDS or ARC patients wereidentified as positive.

The following deposit was made on the date given:

    ______________________________________                                        Strain       Deposit Date                                                                              ATCC No.                                             ______________________________________                                        M13-GAG      January 8, 1986                                                                           40,218                                               ______________________________________                                    

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Pat. Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture for 30 years fromdate of deposit. The organism will be made available by ATCC under theterms of the Budapest Treaty, and subject to an agreement betweenapplicants and ATCC, which assures permanent and unrestrictedavailability of the progeny of the cultures to the public upon issuanceof the pertinent U.S. patent or upon laying open to the public of anyU.S. or foreign patent application, whichever comes first, and assuresavailability of the progeny to one determined by the U.S. Commissionerof Patents and Trademarks to be entitled thereto according to 35 USC§122 and the Commissioner's rules pursuant thereto (including 37 CFR§1.14 with particular reference to 886 OG 638). The assignee of thepresent application agrees that if the culture on deposit should die orbe lost or destroyed when cultivated under suitable conditions, it willbe promptly replaced on notification with a viable specimen of the sameculture. Availability of the deposited strain is not to be construed asa license to practice the invention in contravention of the rightsgranted under the authority of any government in accordance with itspatent laws.

Those skilled in the art should note that the disclosure herein onparticular embodiments of the present invention is exemplary only, andthat various other alternative, adaptations, and modifications may bemade within the scope of the present invention. Accordingly, the presentinvention is not limited to the specific embodiments as illustratedherein, and is embodied in the claims appended thereto.

What is claimed is:
 1. A pair of oligonucleotide primers for apolymerase chain reaction, which primers are sufficiently complementaryto conserved regions among the nucleic acid sequences of AIDS viruses tohybridize therewith and not sufficiently complementary to HTLVI nucleicacids to hybridize therewith, wherein said conserved regions are atleast 20 nucleotides long, wherein said primer pair consists of a memberthat comprises a nucleic acid sequence at least 14 nucleotides inlength, which nucleic acid sequence is contained within a sequenceselected from the group of nucleic acid sequences consistingof5'-ATGAGAGAACCAAGG-3', 5'-CCAGTAGGAGAAAT-3', 5'-ATCCCAGTAGGAGAA-3',and 5'-ATAATCCACCTATCCCAG-3',and a member that comprises a nucleic acidsequence at least 14 nucleotides in length, which nucleic acid sequenceis contained within a sequence selected from the group of nucleic acidsequences consisting of 5'-CCTTGTCTTATGTCCAG-3', and5'-TTATGTCCAGAATGC-3'.
 2. An oligonucleotide primer comprising a nucleicacid sequence which sequence is sufficiently complementary to asubstantially conserved region among the nucleic acid sequences of AIDSviruses, and specific to the nucleic acids of AIDS viruses, to hybridizetherewith and act as a point of initiation of synthesis in anamplification reaction, wherein said primer is selected from the groupconsisting of:5'ATGAGAGAACCAAGG-3', 5'-CCTTGTCTTATGTCCAG-3',5'-CCAGTAGGAGAAAT-3', 5'-ATCCCAGTAGGAGAA-3', 5'-TTATGTCCAGAATGC-3', and5'-ATAATCCACCTATCCCAG-3'.
 3. A DNA probe for detecting or monitoringamplified AIDS virus nucleic acids in a sample capable of hybridizing toan AIDS virus nucleic acid sequence, wherein said probe consists of anucleic acid sequence between 28 and 180 nucleotides in length, whichprobe nucleic acid sequence contains a subsequence at least 28nucleotides in length of a sequence selected from the group of nucleicacid sequences consistingof:5-ATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTAC-3',5-GATCCGAGAGAACCAAGGGGAAGTGACATAGCAGGAACTACTAGTACCCTTCAGGAACAAATAGGATGGATGACAAATAATCCACCTATCCCAGTAGGAGAAATCTATAAAAGATGGATAATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTACCAGCATTCTGGACATAAGACAAGGG-3',and the sequences fully complementary thereto, and wherein said probe,when hybridized to its complementary sequence, encompasses a restrictionenzyme cleavage site.
 4. A DNA probe according to Claim 3 comprising anucleic acid sequence, or a sequence fully complementary to a sequenceselected from the group consistingof:5'-AATCCTGGCCTGTTTAGAAACATCAGAAG-3',5'-TAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGAGAAGCC-3',5-ATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTAC-3', and5'-GATCCGAGAGAACCAAGGGGAAGTGACATAGCAGGAACTACTAGTACCCTTCAGGAACAAATAGGATGGATGACAAATAATCCACCTATCCCAGTAGGAGAAATCTATAAAAGATGGATAATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTACCAGCATTCTGGACATAAGACAAGGG-3'.5. A DNA probe according to claim 3 consisting of a nucleotide sequencebetween 30 and 45 nucleotides long that is fully complementaryto5'-GATCCGAGAGAACCAAGGGGAAGTGACATAGCAGGAACTACTAGTACCCTTCAGGAACAAATAGGATGGATGACAAATAATCCACCTATCCCAGTAGGAGAAATCTATAAAAGATGGATAATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTACCAGCATTCTGGACATAAGACAAGGG-3'or the sequence that is fully complementary thereto.
 6. A DNA probeaccording to claim 5, wherein said restriction enzyme cleavage site is aBstNI site.
 7. A pair of oligonucleotide primers for a polymerase chainreaction consisting of a first and a second primer which aresufficiently complementary to conserved regions among the nucleic acidsequences of AIDS viruses to hybridize therewith and not sufficientlycomplementary to HTLVI nucleic acids to hybridize therewith, whereineach member of said pair of primers consists of a nucleic acid sequenceat least 14 nucleotides in length, wherein said first primer sequence iscontained in the targetsequence5'-GATCCGAGAGAACCAAGGGGAAGTGACATAGCAGGAACTACTAGTACCCTTCAGGAACAAATAGGATGGATGACAAATAATCCACCTATCCCAGTAGGAGAAATCTATAAAAGATGGATAATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTACCAGCATTCTGGACATAAGACAAGGG-3',andwherein said second primer sequence is contained in the sequence that isfully complementary to said target sequence.
 8. A DNA probe fordetecting amplified AIDS virus nucleic acids in a sample, wherein saidprobe is capable of hybridizing to an AIDS virus nucleic acid sequence,and wherein said probe is selected from the group consistingof:5-ATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTAC-3', and5'-GATCCGAGAGAACCAAGGGGAAGTGACATAGCAGGAACTACTAGTACCCTTCAGGAACAAATAGGATGGATGACAAATAATCCACCTATCCCAGTAGGAGAAATCTATAAAAGATGGATAATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTACCAGCATTCTGACATAAGACAAGGG-3'.